Human tissue sulfotransferase (SULT) plays a role for excreting metabolite of lipophilic substrate, to which hydrogen group was introduced by hepatic cytochrome P450 etc., by metamorphosing it into O-sulfate to improve water solubility thereof. SULT is an enzyme group which is classified into a super family and includes gene families such as SULT 1 and SULT 2. It has been reported that, with respect to an enzyme catalyzing sulfoconjugation reaction of phenol substrate that belongs to a SULT 1 family (PSULT), difference occurs in activation thereof according to gene polymorphisms. Further, since this SULT molecular species catalyzes metabolic activation reaction of cancer-causing allylamine, analysis of gene polymorphism is considered to be very important in terms of disease susceptibility. Specifically, among PSULTs, with respect to a molecular species having p-nitrophenol as a representative substrate in human tissue such as liver and platelets (ST1A3), difference in activation occurs according to a polymorphism of the SULT1A1 gene coding for ST1A3. Further, it is known that the character of activation is associated with colon cancer, migraine liability, etc. Among polymorphisms of the SULT1A1 gene, SULT1A1*2 and SULT1A1*3 are strongly associated with disease susceptibility described above. Therefore, analysis of polymorphisms. SULT1A1*2 and SULT1A1*3, with respect to the SULT1A1 gene is very important for predicting disease susceptibility of patients and for preventing and treating them. SULT1A1*2 is a mutation in which arginine (Arg) at position 213 of amino acid is changed to histidine (His) and SULT1A1*3 is a mutation in which methionine (Met) at position 223 of amino acid is changed to valine (Val).
On the other hand, detection of a point mutation, a so-called single nucleotide polymorphism (SNP), is employed widely as a method of analyzing, at the gene level, for example, the causes of all types of diseases and the individual differences in disease liability (susceptibility to diseases) and in drug action. Examples of the common methods of detecting a point mutation include: (1) a direct sequencing method in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by a polymerase chain reaction (PCR) and all the gene sequences are analyzed, (2) a RFLP analysis in which the region corresponding to a sequence to be detected in a target DNA of a sample is amplified by PCR, the amplification product thus obtained is cut with a restriction enzyme whose cleaving action differs depending on the presence or absence of the target mutation in the sequence to be detected and is then electrophoresed, and thereby typing is performed, and (3) the ASP-PCR method in which PCR is performed using a primer with a target mutation located at the 3′-end region and the mutation is judged depending on the presence or absence of amplification.
However, since these methods require, for example, purification of DNA extracted from a sample, electrophoresis, and a treatment with a restriction enzyme, they take time and cost. Furthermore, after PCR is performed, it is necessary to open the reaction container once. Accordingly, there is a possibility that the amplification product may contaminate the next reaction system and thereby the analysis accuracy may be deteriorated. Moreover, since it is difficult to automate, multiple samples cannot be analyzed. Further, the aforementioned ASP-PCR method (3) is less specific, which also is a problem.
Because of these problems, recently, a method of analyzing the melting temperature (Tm) of double-stranded nucleic acid formed of a probe and target nucleic acid is used as a method of detecting a point mutation. Since such a method is performed through, for example, Tm analysis or analysis of the melting curve of the double strand, it is referred to as melting curve analysis. This method is described below. That is, first, a probe complementary to a sequence to be detected containing a target point mutation is used to form a hybrid (double-stranded DNA) between the aforementioned probe and a target single-stranded DNA contained in a detection sample. Subsequently, this hybridization product is heat-treated, and dissociation (melting) of the hybrid accompanying the temperature rise is detected by a change in a signal such as absorbance. The Tm value is then determined based on the result of the detection and the presence or absence of any point mutation is judged accordingly. The higher the homology of the hybridization product, the higher the Tm value, and the lower the homology the lower the Tm value. Therefore the Tm value (reference value for assessment) is determined beforehand with respect to the hybridization product between the sequence to be detected containing a point mutation and a probe complementary thereto, and then the Tm value (measured value) of the hybridization product between the target single-stranded DNA contained in the detection sample and the aforementioned probe is measured. When the measured value is comparable to the reference value, it is considered as matching, that is, it can be judged that a point mutation is present in the target DNA. On the other hand, when the measured value is lower than the reference value, it is considered as mismatching, that is, it can be judged that no point mutation is present in the target DNA. Furthermore, according to this method, it also is possible to automate gene analysis.
However, such a detection method using Tm analysis also has a problem in that a region including a site to be detected must be able to be amplified specifically and efficiently in PCR. Particularly, many isozymes are present in SULT and the sequences for coding them also are very similar to one another. Accordingly, there is a possibility that genes coding for isozymes other than SULT1A1 also are amplified in PCR. Furthermore, when other isozyme-coding genes also have been amplified as described above, it may cause a decrease in reliability of the analysis result in the analysis of, for example, a particular polymorphism (SULT1A1*2 or SULT1A1*3) of the SULT1A1 gene (Nonpatent Document 1 or 2). Moreover, as described above, since analysis of one sample is accompanied by a considerable amount of time and energy it is not practical to analyze multiple samples, which also is a problem.    [Nonpatent Document 1] PMID: 9854023 Biochem J. 1999 Jan. 1; 337 (Pt 1)45-9.    [Nonpatent Document 2] PMID: 9566748 Chem Biol Interact. 1998 Feb. 20; 109 (1-3): 237-48.